Methanotrophs are bacteria capable of using methane as their sole source of carbon and energy. These organisms oxidize methane by the following metabolic pathway:
Microbial ecologists have long been interested in the methanotrophs due to their key role in the global methane cycle, oxidizing CH4 to CO2. The first step in this process is catalyzed by the enzyme methane monooxygenase (MMO), of which two forms are known: the particulate methane oxygenase (pMMO), a membrane-bound enzyme believed to be present in all methanotrophs, and the soluble methane monooxygenase (sMMO), a cytoplasmic enzyme present in only select species. Both forms of the enzyme oxidize a number of compounds, for instance, the common environmental contaminant trichloroethylene. Accordingly, this group of bacteria has attracted interest in regard to their potential for bioremediation.
In some genera of methanotrophs, either sMMO or pMMO is present depending on the copper concentration during growth. At low copper to biomass ratios, sMMO is expressed with low but detectable levels of the pMMO (Choi et al., 2003). At higher copper to biomass ratios, the pMMO is expressed exclusively. The polypeptides and structural genes for both enzymes have been characterized.
In spite of the central role of copper in the physiology of methanotrophs, the mechanism(s) of copper acquisition remains vague. Although true, a few studies have suggested the existence of a specific copper acquisition system in M. capsulatus Bath and M. trichosporium OB3b. The first indication of a specific copper uptake system was provided from phenotypic characterization of the constitutive sMMO mutants (sMMOC) isolated by Phelps et al. (Phelps, P. A., et al. 1992) in Methylosinus trichosporium OB3b mutants having constitutive expression of soluble methane monooxygenase in the presence of high levels of copper. (Appl. Environ. Microbiol. 58:3701–3708). Fitch et al. found that in M. trichosporium OB3b, these sMMOC mutants were defective in copper uptake and showed preliminary evidence for an extracellular copper-complexing agent. Fitch, M. W., et al. 1993. Phenotypic characterization of copper-resistant mutants of Methylosinus trichosporium OB3b. Appl. Environ. Microbiol. 59:2771–2776. Working with the same mutants, Téllez et al. partially purified this copper-complexing agent and determined that it was a small molecule with a molecular mass of approximately 500 Da with an association constant with copper of 1.4×1016M−1. Téllez, C. M., et al. 1998. Isolation of copper biochelates from Methylosinus trichosporium OB3b and sMMOC mutants. Appl. Environ. Microbiol. 64:1115–1122.
Other evidence for a specific copper uptake system was provided by the copper-binding cofactor (CBC) from M. capsulatus Bath. Zahn, J. A., et al. 1996. The membrane-associated methane monooxygenase from Methylococcus capsulatus Bath. J. Bacteriol. 178:1018–1029. During the isolation of the pMMO from M. capsulatus Bath, methanobactin, initially called the copper-binding compound or CBC was identified in association with the purified enzyme, in the washed membrane fraction, and in the extracellular fraction. The CBC was determined to be a small polypeptide with a molecular mass of 1,232 Da. In M. capsulatus Bath, the cellular location of the CBC varied depending on the copper concentration in the culture medium and on the expression of the pMMO.
In a 1998 study, what were believed to be two separate copper-binding compounds (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMOc) mutant, PP319 of Methylosinus trichosporium OB3b. DiSpirito, A. A. et al. (1998), Copper-binding compounds from Methylosinus trichosporium OB3b, 180: 3606–3612. The CBCs were identified as small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L1, and CBC-L2, respectively. The CBC from M. trichosporium OB3b was identical to the CBC previously identified during the isolation of the pMMO from M. capsulatus Bath.
It has now been determined that there is one primary CBC produced by methanotrophs that is involved in copper uptake from the environment. This CBC appears to be the same copper-complexing agent partially purified by Téllez et al. This extracellular molecule has surprisingly been found to have antibiotic and antioxidant properties in addition to copper-chelating properties. For the first time, the CBC has been properly sequenced, and its structural formula identified.
Accordingly, it is a primary objective of the present invention to provide compositions and methods for providing antibiotic activity against using a copper binding compound (methanobactin) that is produced by methanotrophic bacteria.
It is a further objective of the present invention to provide compositions and methods for providing antioxidant activity using methanobactin.
It is still a further objective of the present invention to provide compositions and methods for chelating copper using methanobactin.
The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereafter.